Apparatus for transporting substances into living cells and tissues

ABSTRACT

Inert or biologically active particles are propelled at cell at a speed whereby the particles penetrate the surface of the cells and become incorporated into the interior of the cells. The process can be used to mark cells or tissue or to biochemically affect tissues or tissue in situ as well as single cells in vitro. Apparatus for propelling the particles toward target cells or tissues are also disclosed. A method for releasing particles adhered to a rotor device is also disclosed.

This is a continuation of application Ser. No. 07/074,652 (abandoned)filed Jul. 17, 1987, which is a divisional application of applicationSer. No. 06/670,771 filed Nov. 13, 1984, now U.S. Pat. No. 4,945,050.

The present invention relates to a novel method and apparatus fortransporting substances into living cells and tissues without killingthe cells and tissues.

BACKGROUND OF THE INVENTION

Biologists often need to introduce into living cells a wide range ofsubstances which are normally excluded from the cell by cell walls andouter cell membranes. Such substances include biological stains,proteins, nucleic acids, organelles, chromosomes, and nuclei.

The importance of introducing biological substances into cells isreflected by the large amount of work which has been done in this area,and the expensive technologies which have been developed to achieve thisend. While diverse applications of biological delivery systems are known(Introduction of Macromolecules into Viable Mammalian Cells, (Ed. R.Baserga, C. Crose, G. Rovera), Winstar Symposium Series VI, 1980, A.R.Liss Inc., New York), one application of central importance will clearlybe the introduction of genetic material into cells for the purpose ofgenetic engineering. Existing technologies for transporting geneticmaterial into living cells involve uptake mechanisms, fusion mechanisms,and microinjection mechanisms.

Uptake mechanisms include: (a) induction of enhanced membranepermeability by use of Ca⁺⁺ and temperature shock (Mandel, M. and Higa,A., 1970, "Calcium Dependent Bacteriophage DNA Infection," J. Mol.Biol., 53: 159-162; Dityatkin, S. Y., Lisovskaya, K. V., Panzhava, N.N., Iliashenko, B. N., 1972, "Frozen-thawed Bacteria as Recipients ofIsolated Coliphage DNA", Biochimica et Biophysica Acta, 281: 319-323);(b) use of surface binding agents such as polyethylene glycol (PEG)(Chang, S. and Cohen, S. N., 1979, "High Frequency Transformation ofBacillus subtilis Protoplasts by Plasmid DNA", Mol. Gen. Genet., 168:111-115; Krens, F. A., Molendijk, L., Wullens, G. J., and Schilperoort,R. A., 1982, "In vitro Transformation of Plant Protoplasts withTi-Plasmid DNA", Nature, 296: 72), or Ca(PO₄)₂ (Graham, F. L., and vander Eb, A. J., 1973, "A New Technique for the Assay of Infectivity ofHuman Adenovirus 5 DNA", Virology, 52: 456; Wigler, M., Sweet, R., Sim,G. K. Wold, B., Pellicer, A., Lacey, E., Maniatis, T., Silverstein, S.,and Axel, R., 1979, "Transformation of Mammalian Cells with Genes fromProcaryotes and Eucaryotes", Cell 16: 777); and (c) phagocytosis ofparticles such as liposomes (Uchimiya, H., Ohgawara, T., and Harada, H.,1982, "Liposome-mediated Transfer of Plasmid DNA into PlantProtoplasts", In: Fujiwara A. (ed.), Proc. 5th Intl. Cong. Plant Tissueand Cell Culture, Jap. Assoc. for Plant Tissue Culture, Tokyo, pp.507-508), organelles (Potrykus, I., 1973, "Transplantation ofChloroplasts into Protoplasts of Petunia", Z. Pflanzenphysiol., 70:364-366), or bacteria (Cocking, E. C., 1972, "Plant Cell ProtoplastsIsolation and Development", Ann. Rev. Plant Physio., 23: 29-50), intothe cell. These uptake mechanisms generally involve suspensions ofsingle cells, from which any existing cell wall materials have beenremoved enzymatically.

Uptake protocols are generally quite simple, and allow treatment oflarge numbers of cells en masse. However, such methods tend to have verylow efficiency. In plant protoplasts, transformation frequencies tend tobe one in 10,000 or less, while in animal cell uptake systems,transformation frequencies tend to be even lower. In such systems mostcells are unaffected, and special cell selection procedures are requiredto recover the rare cells which have been transformed (Power, J. B. andCocking, E. C., 1977, "Selection Systems for Somatic Hybrids", In:Reinert, J. and Bajaj, Y.P.S. (eds.) Plant Cell, Tissue, and OrganCulture, Springer-Verlag, N.Y., pp. 497-505).

Fusion mechanisms incorporate new genetic material into a cell byallowing one cell to fuse with another cell. A variation on thisprocedure involves "ghost" cells. The membrane of killed cells areallowed to fill with a given DNA solution, such that cell fusionincorporates the DNA from the carrier "cell" into the living cell.Cell-to-cell fusion can be induced with the aid of substances such asPEG (Bajaj, Y.P.S., 1982, "Protoplast Isolation, Culture and SomaticHybridization", In: Reinert, J. and Bajaj, Y.P.S. (eds.) Plant Cell,Tissue, and Organ Culture, Springer-Verlag, N.Y. pp. 467-496), andSendai virus particles (Uchida, T., Yamaizumi, M., Mekada, E., Okada,Y., 1980, "Methods Using HVJ (Sendai Virus) for Introducing Substancesinto Mammalian Cells:, In: Introduction of Macromolcules into ViableMammalian Cells, Windsor Symposium Series V.I. A.R. Liss Inc., N.Y., pp.169-185).

As with uptake mechanisms, fusion technologies rely upon the use ofsingle cell suspensions, where cells are enzymatically stripped of anycell wall material. While fusion technologies can have relatively goodefficiencies in terms of numbers of cells affected, the problems of cellselection can be more complex, and the resulting cells are typically ofelevated ploidy, which can limit their usefulness.

Microinjection technologies employ extremely fine, drawn out capillarytubes, which are sometimes called micropipettes. These capillary tubescan be made sufficiently small to be used as syringe needles for thedirect injection of biological substances into certain types ofindividual cells (Diacumakos, E. G., 1973, "Methods for Microinjectionof Human Somatic Cells in Culture", In: Prescott DM (ed.) Methods inCell Biology, Academic Press, N.Y. pp. 287-311; Graessman, M. andGraessman, A., 1983, "Microinjection of Tissue Culture Cells", Methodsin Enzymology, 101: 482-492). When small cells need to be injected, verysharp microelectrodes are required, whose tips are very easily broken orclogged. Very high pressures are required to cause bulk flow throughorifices smaller than one micron. Regulation of such bulk flow is verydifficult. The entire process is something of an art, requiringdifferent modifications for different cell types. One modification ofmicroinjection involves pricking with a solid-glass drawn needle, whichcarries in biological solutions which are bathing the cell (Yamamoto,F., Furusawa, M., Furusawa, I., and Obinata, M., 1982, "The "Pricking"Method", Exp. Cell Res., 142: 79-84). Another modification, calledionophoresis (Purres, R. D., 1981, Microelectrode Methods forIntracellular Recording and Ionophoresis, Academic Press, N.Y., p. 146;Ocho, M., Nakai, S., Tasaka, K., Watanabe, S., and Oda, T., 1981,"Micro-injection of Nucleic Acids Into Cultured Mammalian Cells byElectrophoresis", Acta Med. Okayama, 35(5): 381-384), useselectrophoresis of substances out of the microelectrode and into thecell, as an alternative to high pressure bulk flow.

Microinjection procedures can give extremely high efficiencies relativeto delivery into the cell. Because of this, microinjection has been usedsuccessfully in the transformation of individual egg cells. Onedisadvantage of microinjection is that it requires single cellmanipulations and is therefore inappropriate for treating masses ofcells and is generally a very tedious and difficult technology.Microinjection is a technology which would not be easily universalizedor automated.

While a variety of biological delivery systems presently exist, none ofthese technologies is free from major limitations. Perhaps the greatestsingle limitation of all of these technologies is that they are limitedto single cell in vitro systems.

SUMMARY OF THE INVENTION

Accordingly, one object of the present invention is to provide a widelyapplicable mechanism for transporting particles, which can comprise orbe associated with biological substances, into living cells whichmechanism does not depend upon the cell type, size, shape, presence orabsence of cell wall, cell number, or cellular environment.

Another object of the present invention is to provide a mechanism fortransforming large numbers of cells simultaneously and is not limited tosingle cell manipulation.

A further object of the present invention is to provide a mechanism foraffecting tissues in situ as well as single cells in vitro.

These and other objects can be achieved by increasing the kinetic energyof particles which can comprise or act as carriers for the biologicalsubstance sought to be inserted in cells, and propelling the particlesat the cells at a speed whereby the particles penetrate the surface ofthe cells and become incorporated into the interior of the cells.

Broadly, these objectives can be accomplished by apparatus adapted toaccelerate the particles to a predetermined speed and further adapted topropel the particles toward a target, preferably in a directed manner.One such apparatus, which can be referred to as a "gas blast" device,comprises tube means closed at one end and having an outlet at the otherend, means for injecting a pressurized gas into said tube means adjacentthe closed end thereof, apertured deflector means adjacent the other endof the tube means for deflecting a portion of the pressurized gas toprevent undue damage to the cells, and delivery means communicating withthe tube means intermediate the ends thereof for supplying particlesinto the tube means in a manner adapted to entrain the particles in thepressurized gas passing throug the tube means.

Another means for accelerating the particles comprises a macroprojectilecontaining the particles, means for accelerating the macroprojectile andmeans for stopping the macroprojectile while allowing the particles tomaintain the previously acquired velocity and to be propelled toward atarget.

Yet a third means for accelerating the particles to high velocitiescomprises a high speed rotational device which accelerates to a desiredvelocity, particles bound or dispersed to its outer perimeter andreleases the particles in a manner which propels at least a portion ofthe particles toward a target. Preferably, the rotational devise isoperably associated with a means which releases at least a substantialportion of the particles in a predetermined straight path tangential tothe localized point of release.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1a to 1d show four methods of introducing biological substancesinto cells by the use of accelerated particles. In 1a, an inertmicrosphere is coated with a biological substance, accelerated, andafter penetrating the target cell microsphere releases the substancefrom its surface. In 1b, a target cell is surrounded with a biologicalsubstance, and is bombarded with an uncoated inert microsphere whichcarries the biological substance into the cell in its wake. In 1c, adried bacterium is employed as a projectile, carrying DNA in itsinterior, which is released into the target cell after penetration andbacterial lysis. In 1d, a bacteriophage particle is used as aprojectile, carrying DNA in its interior which is released into thetarget cell after penetration and phage coat breakdown.

FIGS. 2a to 2e show various mechanisms for acceleration of smallparticles to high velocity, for the purpose of bombarding cells. In 2a,compressed gas is used to accelerate particles simply and directly. In2b, a blast-plate is used to translate the kinetic energy of abullet-sized particle to the small particles on the far side of a plate.This offers potentially higher velocities with less associated airblast. In 2c, a bullet-sized particle is used to accelerate smallparticles in a forward cavity, and a stopping device is used to stop thelarger particle, while allowing the small particles to continue fullspeed. In 2d, a high-speed rotor is used to accelerate to very highvelocities small particles attached or dispersed to its perimeter. Whenallowed to escape from the rotor, such particles can come off the rotortangentially at high speed. In 2e, electrostatic acceleration of smallcharged particles is employed as is used in sub-atomic accelerators.

FIGS. 3a and 3b show the introduction of four-micrometer diametertungsten spheres into onion epidermal cells using particle accelerationas depicted in FIG. 2a. FIG. 3a shows the exterior of the cell coveredwith tungsten spheres following bombardment. FIG. 3b shows tungstenspheres having entered the interior of the same cells (focusing 15micrometers below the cell surface).

FIGS. 4a and 4b show the introduction of four-micrometer diametertungsten spheres into onion epidermal cells using particle acceleration.FIG. 4a shows the exterior of the cell covered with tungsten spheresafter bombardment. FIG. 4b shows tungsten spheres which have entered theinterior of the same cells (focusing 50 micrometers below the cellsurface).

FIGS. 5a and 5b show onion epidermal cells after bombardment withfour-micrometer diameter tungsten spheres travelling at velocities of afew hundred feet per second. FIG. 5a shows a cell (indicated by thearrow) having five tungsten spheres on the surface. FIG. 5b shows thesame cell with a focus at about 65 micrometers beneath the cell surface.

FIGS. 6a and 6b show onion epidermal cells after bombardment withfour-micrometer diameter tungsten spheres travelling at velocities of afew hundred feet per second. FIG. 6a shows 16 tungsten spheres on thesurface of the cell. FIG. 6b shows nine tungsten spheres in the interiorof the same cell with a focus at about 65 micrometers beneath the cellsurface.

FIG. 7a is a schematic diagram of a compressed gas accelerator accordingto the present invention with one embodiment of a deflector. FIG. 7b isa schematic diagram of another embodiment of a deflector.

FIG. 8a is a schematic diagram of a accelerator apparatus whichaccelerates a macroprojectile containing particles and a means forstopping the macroprojectile while allowing the particles to proceedtoward a target with a predetermined acquired velocity.

FIG. 8b is a diagram of a macroprojectile containing particles.

FIG. 9 is a schematic diagram of an accelerator apparatus employingcentripetal acceleration. Particles are adhered to the outer surface ofa rotational device and the device accelerated to a predetermined speed.The particles are freed from the surface of the rotating device forexample by means of a directed energetic beam, e.g. a laser beam, whichallows the particles to continue at a predetermined velocity in astraight line tangential to the point of release.

FIG. 10 is a photograph of the pattern of tungsten spheres as releasedfrom a high speed rotational device by a YAG laser beam, as collected onScotch tape 3 inches from the point of release. The actual beam isround, but the pattern on the tape is oblong because the tape was at a45° angle to the particle beam.

FIG. 11 is a microphotograph of the same pattern of tungsten spheres asin FIG. 10, showing the absence of clumping and the uniform dispersionof the particles.

DETAILED DESCRIPTION OF THE INVENTION

According to the method of the present invention, particles of theappropriate size, accelerated to appropriate velocities can readilypenetrate thin barriers such as cell walls and cell membranes, therebyentering into the cell cytoplasm. In this way, particles such as inertparticles coated, impregnated, or otherwise operably associated withbiological substances, or frozen or otherwise solid biological particlescan be introduced into cells FIGS. 1a to 1d.

The only physical limitation upon the particles is that they havesufficient mass to acquire the necessary kinetic energy to penetrate theparticular cell sought to be penetrated and that they have integritysufficient to withstand the physical forces inherent in process.

The size of the particles is only broadly critical. Usually theparticles have a diameter between about 10 nanometers and about a fewmicrometers. In order to penetrate the cell and become incorporated intothe interior of the cell without killing the cell, the maximum size ofthe particle must be a size substantially smaller than the cell soughtto be penetrated. Small cells e.g. cells with a diameter of about tenmicrometers or less usually will only tolerate particles having adiameter about ten times smaller than their own diameter. Larger cellstend to tolerate particles having larger particle diameter. Theviability of cells depends in part on the particular cells and theenvironment of the cell at the time of penetration. Where necessary themaximum size of particle tolerated by a particular cell can be readilydetermined by accelerating inert particles into cell samples andexamining the viability of the resultant cell samples. The minimum sizeof the particle is governed by the ability to impart sufficient kineticenergy to penetrate the desired cell. Generally, the optimum particle issmall enough to produce minimal cell damage and large enough to acquiresufficient momentum to penetrate the cell; momentum being a function ofsize, density and velocity.

The velocity to which the particles must be accelerated likewise dependson the size and density of the particle, as well as the nature of thephysical barriers surrounding the particular cell. The desired velocityis that minimum velocity sufficient to impart the required kineticenergy to cause the particle to penetrate and become incorporated into adesired cell. For onion epidermal cells the velocity required for cellpenetration by 4 micrometer tungsten spheres is in the order of about400 feet per second. The velocity for cells with thinner cell walls orno cell walls will be a function of particle size and density andrequired penetration depth. As previously stated the particle velocityrequired for a particular cell in a particular environment can bedetermined by the use of inert spheres of appropriate size and density,e.g. metal or plastics or mixtures thereof, prior to the use ofparticles comprising the biological material.

According to the method of the present invention, the particles can beaccelerated by any accelerating means suitable for accelerating smallparticles. The accelerating means is not critical, provided that themeans is capable of providing a plurality of particles to a specifictarget at a predetermined velocity in a manner which does not adverselyeffect the biological substance associated with the particle. Examplesof such accelerating means include gas blast means (e.g. FIG. 2a),mechanical impulse means (e.g. FIGS. 2b and 2c), centripetal means (e.g.FIG. 2d) and electrostatic means (e.g. FIG. 2e). Within the scope ofthis invention the method of the invention can be practiced byaccelerating means which operate on the above principles or otherprinciples which accomplish the desired result. The structural detailsof any specific apparatus can vary from these specifically discussedherein as can be perceived by one skilled in the art of accelerationdevises.

As set forth above, the particles can be, for example, inert particles,particles coated or impregnated with biological substances, or frozen orotherwise solid biological particles.

Examples of inert particles include ferrite crystals, gold or tungstenspheres, and other metal spheres and particles of high density, forexample about 10 to about 20 g/cm³ as well as spheres and particles oflow density (for example 1-2 gm/cm³) such as glass, polystyrene, andlatex beads.

Biological particles include any biological substance which can befreeze dried, or otherwise prepared as free particles or otherwise usedas a particle projectile for cell penetration. Examples of suchparticles include bacteria, viruses, organelles, and vesicles. Once inthe cells, such biological particles or portions thereof would beexpected to return to their natural state undamaged (e.g. hydrate, thaw,dissolve, etc.) or otherwise to contribute a desired biological activitywithin the cell.

Further, according to the present invention, biological substances canbe coated on, bonded on or precipitated onto the surface of inertparticles such as latex beads, high density metal spheres or ferritecrystals, or the particles can be impregnated with various biologicalsubstances. Such coated or impregnated particles can then act ascarriers, carrying the biologically active substances into the cell.Once in the aqueous environment of the cytoplasm, the biologicalsubstances would dissolve or be dispersed into the cyto-solution (FIG.1a).

Additionally, the cells can be bathed in or surrounded by a biologicalsolution and bombarded with inert particles to pull into the cell, inthe wake of the particles, a given volume of the external biologicalsolution (FIG. 1b). The particles can be uncoated or coated with abiological substance which is the same or different from the biologicalsubstance bathing or surrounding the cells. In the same manner,biological particles can be propelled at cells bathed in or surroundedby a biological solution.

Examples of biological substances which can be coated onto orimpregnated into inert particles or used to bathe the cell includebiological stains such as fluorescent or radiolabeled probes, viruses,organelles, vesicles, proteins such as enzymes or hormones, and nucleicacids such as DNA and RNA.

Such penetration of living cells with small particles projected from aparticle accelerator is possible with a minimum of cell handling, cellpreparation, or cell disruption. Lesions in the cell membrane need notbe much larger than would be achieved using microinjection proceduresand need only remain open for a fraction of a second, e.g., thetransient time of the particle. Particles can be accelerated in largenumbers to affect large numbers of cells simultaneously.

Further, according to the present invention, the cell type, size, shape,presence or absence of cell wall, cell number, or cellular environmentis not critical and should not significantly alter effectiveness.Examples of the wide array of cells which can be subjected to thisinvention include algal cells, bacteria, single cell protozoa, plantpollen, plant protoplasts, animal eggs, animal bone marrow cells, muscleor epidermal cells, or any similar plant or animal cell.

Additionally, since there is spacial separation between the transformingapparatus of the present invention and the recipient cells, the presentinvention allows for the treatment or modification of cells in tissuesin their natural state, i.e., in situ. Examples of tissues which can bebombarded include plant tissue such as meristem tissue, and human tissueor other animal tissue such as epidermal tissue, organ tissue, tumortissue and the like. It is noted that air-delivered injection devicesare currently employed medically to administer vaccines, but theadministration is into tissue fluid or blood, and not directly intoliving cells. Such tissue treatment or modification would require suchlevels of particle bombardment of a tissue which would not be lethal totissue, although some cells might die, but which would affect asignificant fraction of the cells in that tissue.

One embodiment of the apparatus of the present invention isschematically illustrated in FIG. 7a and is basically comprised of aparticle accelerator device 10 and a blast deflector 12. The particleaccelerator 10 is comprised of an elongated hollow accelerator tube 14having an end wall 16 closing one end of the tube. A source ofcompressed gas 18 communicates with the accelerator tube 14 adjacent theclosed end thereof by means of the passage 20 which is controlled by asuitable valve means 22 adapted to be selectively controlled by anoperator. A pair of particle inlets 24 and 26 are provided in spacedrelation to each other along the length of the tube 14. As illustratedin FIG. 7a, a suitable particle supply means 28 is threaded into theinlet 24 adjacent the closed end of the tube and a plug 30, in the formof a screw or the like, is threaded into the inlet 26. Depending uponthe nature of the particles involved and the desired acceleration forthe particles, the particle supply means and the plug 30 can be reversedin the inlets 24 and 26.

In the operation of the apparatus, the valve 22 is operated for adiscrete period of time, thereby allowing a blast of compressed gas toenter into the closed end of the tube 14. As the blast of compressed gastravels along the length of the tube toward the outlet 32, a supply ofparticles from the particle supply device 28 will be delivered into thegas stream through the inlet 24. The stream of gas carrying theparticles will then impinge upon a cell substrate disposed at anappropriate distance from the outlet 32 of the tube 14.

In the event that it is necessary to locate the cell substrate inextremely close proximity to the outlet 32 of the accelerator tube 14, ablast deflector 12 may be provided to prevent undue cell damage. Theblast deflector 12, as illustrated in FIG. 7a, is comprised of a plate34 having an aperture 36 extending therethrough with a reticulatedscreen 38 secured in the aperture 36. The blast deflector 12 may befitted over the end of the accelerator tube 14 with the screen 38aligned with the outlet 32. Suitable exhaust apertures 40 may beprovided in the end of the tube 14 adjacent the outlet 32 so that themain blast may be deflected laterally outwardly through the apertures 40while permitting a small amount of the airstream with the particlesentrained therein to pass through the reticulated screen 38 forimpingement on the cell substrate.

A modified form of the blast deflector 12' is shown in FIG. 7b and iscomprised of two interconnected oppositely directed truncated conicalmembers 42 and 44. The truncated conical member 42 is open at both endswith the smaller diameter opening 46 adapted to be disposed adjacent theoutlet 32 of the accelerator tube 14. Any suitable means can be providedfor mounting the blast deflector 12' adjacent the end of the accelerator14. Thus, a small amount of the air stream with the particles entrainedtherein can pass through the small diameter opening 46, while the mainblast is deflected along the outer conical surface of the member 42. Themain blast will then impinge upon the conical surface of the oppositelydirected member 44 to deflect the blast laterally outwardly andrearwardly away from the cell substrate.

The details of the above particle accelerator and the blast deflectormay be varied within the scope of the present invention.

Another embodiment of the apparatus of the present invention isillustrated in FIG. 8a and is comprised of a macroprojectile 50 (FIG.8b), with a forward cavity 53 containing particles 52, contained in tubemeans 54, which has a discharge means 55, for example an explosivecharge or compressed gas source rearward the macroprojectile, e.g.mounted at rear end 56 of the tube means, which discharge, means isactuated by a triggering means 57. The forward end of the tube means hasa macroprojectile stopping means 58 adapted to stop the acceleratedmacroprojectile while allowing the particles to continue through aaperture 59, toward a target cell or tissue 60. Vent means, e.g. holes,61 in the tube means allows dissipation of the accelerating forceproximal the forward travel limit of the macroprojectile. To minimizeair resistance the entire apparatus and target can be enclosed in avacuum chamber 57 connected to a vacuum pump 62, to allow the process tobe operated at reduced air pressure.

Yet another embodiment of the apparatus of the present invention isillustrated in FIG. 9. Particles 70 are bound or adhered to the outeredge of a rotor 71 and thereby accelerated to a high velocity. Thevelocity of the particles is controlled by the circular velocity of therotor. Controlled release of the particles is achieved by focusing anenergetic beam 73, such as a laser beam, at a specific point on theouter edge of the rotor. The energy of the beam is adapted to and causesrelease of particles from the surface of the rotor, causing suchparticles 74 to continue at a predetermine velocity in a straight linetangential to the point of release and thus to be directed toward atarget. This result can optimately be achieved in a vacuum chamber 75which allows maximal speeds for the rotor and minimal frictionaldeacceleration of the particles. Preferably, the target cell or tissue76 is placed in a sample compartment 77, separable from the main vacuumchamber 75 by a movable airlock door 78 and having independent means forcontrol of the pressure to allow changing of samples without loss ofvacuum in the main chamber. This preferred two chamber embodiment alsoavoids extended sample exposure to hard or sudden vacuum which wouldresult in tissue desiccation. The sample chamber can be fitted with asample viewing port 79, or alternatively, with a opening surrounding bya sealing means (not shown) to allow the use of a human or animalanatomical portion or a portion of a whole living plant as a target.

It is significant to note that the use of an energetic beam such as alaser beam, ion beam, electron beam to dislodge particles adhered orbound to a rotor means to cause particles to flow in a path tangentialthe rotor arc is considered novel. The energy beam is selected to impartenergy of a nature and in an amount sufficient to overcome the forces ofadhesion existing between the particle and the rotor means and releasethe particle from the rotor without adversely affecting the particles'essential properties.

An alternative embodiment of the above centripetal device comprises arotor having one or more passageways from its center to its outer edgeadapted to dispense particles from the outer edge of the rotor. In thisdevice particles would be dispensed over a 360° arc. This apparatuswould be less efficient with respect to a small target, but on the otherhand would allow the use of multiple or larger area targets mounted onthe inside wall of an enclosure surrounding the rotor.

As is clear from the above detailed description of the presentinvention, the nature of the accelerated particle or "particle gun"approach is such that it should be a suitable substitute for anyexisting intracellular delivery system. The potential applications areextremely diverse. Moreover, a particle accelerator has the new andnovel capability to transform general tissues in situ. This new andnovel capability gives rise to new and novel potential applications ofthe technology, not possible with any other technology. Two importantnew applications of particle accelerator mediated in situ tissuetransformation are considered to be: (1) germline transformation ofplants, and (2) human gene therapy. These new applications aredemonstrated below and are illustrative only and not intended to limitthe present invention.

Germline Transformation of Plants

Most plant species cannot currently be regenerated from protoplasts.Even in those species which can be regenerated, the time and expenseinvolved in regeneration represent significant obstacles. There has beenconsiderable effort devoted to the problem of regeneration in many plantspecies, often without success. To continue to try to develop reliableregeneration procedures for each crop plant will be very expensive, andin many cases prove impractical or impossible. These problems can becompletely circumvented by using germline cells instead of plantprotoplasts, as targets for genetic manipulation.

The two logical targets for plant germline transformation are pollen andmeristem domes. Transformation of pollen is the method of choice forsexually-propagated crops, such as grain crops while transformation ofmeristematic domes is the method of choice for asexually-propogatedcrops such as most fruits, potato, sugar cane, etc. Either approachwould be expected to directly produce transformed whole plants.

While it is conceivable that plant pollen can be effectivelymicroinjected, particle bombardment would be much more effective, sincethousands of pollen cells could be treated simultaneously. Meristemtransformation could only be achieved by particle bombardment asdescribed below.

The meristematic dome can be exposed surgically, as is commonly done formeristem culturing and then bombarded with DNA-carrying particles.Exposure times could be increased up to a near-lethal level, asdetermined emperically. At this point a large number of meristematiccells can be transformed. Treated meristems are then allowed to grow outinto chimeric shoots, from which stable transformed sectors areselected.

Somatic Human Gene Therapy

The potential significance of human gene therapy has been recognized forquite some time (The Prospects of Gene Therapy, 1971, FogartyInternational Center Conference Report, Fresse E. (ed.), DHEW Publ. No.(NIH 72-61)), although this has not been a particularly active area ofresearch for lack of effective means of incorporating genes into humantissues. Over 1500 human diseases are known to be genetically determined(McKusick, V. A., 1971, Mendelian Inheritance in Man, John HopkinsPress, Baltimore). There are at least 92 human disorders for which asingle enzyme deficiency is the cause, i.e., based upon a single genedefect (McKusick, V. A., 1970, Ann. Rev. Genet., 4:1). When geneticdisease is associated with an enzyme deficiency in a specific tissue,particle gun bombardment can be used to introduce therapeutic genes.

For example, a patient with sickle cell anemia can have bone marrowtissues bombarded with particles carrying the appropriate DNA, i.e.inserting sequences expressing functional Hb^(a) gene, to restore normalfunction to those tissues, producing a sufficient number of normal redblood cells to produce a healthy individual.

Healthy tissues can likewise be subjected to particle bombardmenttherapy, where significant improvements might be made.Disease-resistance conferring genes might potentially be engineered forexample, by using parasite-derived resistance as described by Sanfordand Johnston (in press). Alternatively, health-prompting biosyntheticcapabilities might be introduced in human tissues, such as ability tosynthesize vitamins, or new anticancer compounds (Ames B. N., 1983,"Dietary Carcinogens and Anticarcinogens", Science, 221:1256-1264).

While the emphasis herein has been placed on the use of the particles toadd biological material to a cell, it should be noted that the insertionof biological inert particles into cells is within the scope of theinvention and has utility. The particles can be used, for example, ascell markers and the like.

The present invention will now be described by reference to specificexamples which are meant to be illustrative only and are not intended tolimit the invention.

EXAMPLE 1 Demonstration Of Penetration Of Cell By Metal SpheresAccelerated By Gas Blast Apparatus

Onion epidermal cells were bombarded with tungsten spheres one and fourmicrometer in diameter, which had been accelerated to a high velocityusing an air blast generated by the apparatus described above. The outerscales of a mature onion were removed to expose younger scales. Theexpressed epidermis of such inner scales were bombarded with no furtherpreparation. The distance between the particle accelerator and theepidermal cells varied from 1 cm to 20 cm, with greater distanceyielding less cellular disruption, but lower percent penetration. Theexit velocity of the air blast from the accelerator was estimated to bebelow about 600 feet per second.

Both the one-micrometer and four-micrometer spheres entered into theonion epidermal cells without any gross damage resulting to the surfaceof the epidermal cells.

A fraction (about 5%) of the one-micron tungsten spheres penetrated theonion epidermal cells. To achieve a higher rate of penetration withsmaller particles, a higher velocity can be used.

Results of the bombardment with four-micrometer spheres are shown inFIGS. 3a, 3b, 4a, 4b, 5a, 5b, 6a, 6b. As can be seen from the figures,four-micrometer spheres entered into onion epidermal cells insignificant numbers. Rates of particle penetration in some casesapproached 50% of the spheres employed. Further, large numbers of cellswere simultaneously penetrated, often with multiple particles. Theresults also indicate that accelerated tungsten spheres penetrate asdeeply as three cell layers.

As can be seen from FIG. 3b (focusing 15 micrometers below the cellsurface), four-micrometer diameter tungsten spheres penetrated the cellmembrane and entered the interior of the cell with no obvious or grosslesions appearing on the cell surface.

FIG. 4b shows four-micrometer diameter tungsten spheres introduced intoonion epidermal cells (at a focus of 50 micrometers below the cellsurface). The sphere in the center (arrow) created a star-shapeddepression on the far-side envelope of the cell, indicating that theparticle still had measurable momentum after passing into the cell.

FIGS. 5a and 5b show onion epidermal cells which have been bombardedwith four-micrometer diameter tungsten spheres travelling at velocitiesof a few hundred feet per second. FIG. 5b shows spheres in the interiorof the cell (at a focus of about 65 micrometers beneath the surface ofthe cell). As can be seen, there was no apparent cell disruption.

FIGS. 6a and 6b also show onion epidermal cells after bombardment withfour-micrometer diameter tungsten spheres travelling at velocities of afew hundred feet per second, but at a higher magnification than shown inFIGS. 5a and 5b (4-micrometer spheres give scale). As can be seen fromFIG. 6b nine tungsten spheres entered the interior of the cell (focuswas at about 65 micrometers beneath the surface of the cell). Again, noapparent cell disruption was observed.

EXAMPLE 2 Demonstration Of Cell Viability After Penetration By MetalSpheres

Onion epidermal cells were evaluated relative to their viabilityfollowing bombardment with four-micrometer tungsten spheres, using theconditions of bombardment set forth in Example 1. Although the air blastkilled many of the surface cells, a significant number of cells remainedalive as determined by microscopic observation of cytoplasm streaming.The number of cells killed was largely a function of the distance of thecells from the accelerator device. The cells containing tungsten sphereshad very active cytoplasmic streaming, both one hour after bombardmentand 20 hours after bombardment. In some cases, a tungsten sphere couldbe seen carried along within the cell by the cytostreaming.

EXAMPLE 3 Demonstration Of Mechanical Impulse Acceleration Of Particles

This method for accelerating small particles is based upon the conceptof mounting the small particles on a larger particle or surface,accelerating the larger body by impact or ballistic means, and thenstopping the larger body, while allowing the smaller particles tomaintain their velocity (see FIGS. 2b and 2c). With reference to FIG.2b, a high mass projectile (a lead pellet) was accelerated toapproximately 600 feet per second by conventional means (a commercialpellet gun). This was used to impact against a 0.8 mm thick copperplate. Adhering to the reverse side of the plate were 4-micrometertungsten spheres. As the plate was deformed by the impact, the sphereswere accelerated to a velocity similar to that of the lead pellet, andwhen the copper plate reached the limit of its deformation, the tungstenspheres separated from its surface and continued at highvelocity-penetrating onion epidermal cells in a manner very similar tothat observed using air blast acceleration. The area of effectivepenetration was smaller than using an air blast, being limited to thearea immediately below the pellet's point of impact on the copper plate.This method is desirable in that higher velocities than with an airblast should be attainable, and in the absence of the potentiallydamaging air blast. A modification of this principle is represented inFIG. 2c. A scale up design of an apparatus employing this principle isillustrated in FIG. 82.

EXAMPLE 4 Demonstration Of Centripetal Acceleration Of Particles

This method exploits the high velocities generated on the perimeter of ahigh speed rotor, for the purpose of accelerating small particles (FIG.2d). Using ultracentrifuge technologies, outer perimeter velocities onrotors can reach several thousand feet per second. Particles could bedelivered to the outside of the rotor by a variety of mechanisms,including channels radiating from the center of the rotor, or by astream of particles to be caught by external ridges on the outer surfaceof rotor.

With reference to FIG. 9, four micrometer tungsten spheres were adheredto the rotor of an Eppindorff microcentrofuge by applying the particlesas an ethanol suspension, followed by drying. A pulsed YAG laser wasfocused on the surface of the spinning rotor, such that particles werereleased from the surface of the rotor and flew tangentially from thepoint of laser contact. The particles were collected on Scotch tape, todetermine the width of the resulting particle beam. In spite of high airturbulence (no vacuum was employed), the beam was relatively tightlyfocused, being only 2 and 3 times the width of the laser beam (3 mm), 3inches from the point of release (see FIG. 10). The pattern ofdistribution within the particle beam was examined microscopically, andwas found to be extremely uniform with no clumping (FIG. 11). The rotorhad a circumference of 22 inches and a speed greater than 12,000 roundsper minute, giving the particles a velocity greater than 366 feet persecond. The YAG laser was used at a frequency of 10 pulses per second.Wavelengths of 532 nm and 1.06 um were both found to be effective inreleasing the particles, when used in the energy range of 15-30millijoules.

Particles have been similarly attached to an ultracentrifuge rotor andhave been brought us to velocities as high as 1,600 feet per second,greatly exceeding those velocities found to be effective for cellpenetration using the two previous methods described above. Thisapparatus is capable of delivering particles over a wide range ofspeeds.

While the invention has been described in detail and with reference tospecific embodiments thereof, it will be apparent to one skilled in theart that various changes and modifications can be made therein withoutdeparting from the spirit and scope thereof.

We claim:
 1. An apparatus for introducing one or more particles into atleast one cell which comprises said one or more particles associatedwith means to accelerate said one or more particles to thereby causesaid one or more particles to penetrate the at least one cell andwhereby at least a portion of said one or more particles becomesincorporated into the interior of the at least one cell, wherein saidone or more particles comprise biological substances and have a diametersufficiently small to penetrate the at least one cell without killingthe at least one cell.
 2. The apparatus as set forth in claim 1 furthercomprising means associated with said means to accelerate in order todirect the accelerated one or more particles toward the at least onecell.
 3. An apparatus as in claim 1, wherein said means to acceleratecomprises discharge means having a periphery and a solid substratecarrying said one or more particles, wherein said discharge meansimparts momentum to said solid substrate carrying said one or moreparticles which in turn accelerates said one or more particles.
 4. Anapparatus as in claim 3, wherein said solid substrate comprises amacroprojectile carrying said one or more particles, and said apparatusfurther comprises a macroprojectile stopping means, located contiguousthe periphery, to stop said macroprojectile while allowing said one ormore particles to continue toward the at least one cell.
 5. An apparatusas in claim 3, further comprising a substrate stopping means locatedcontiguous the periphery, to stop said substrate carrying said one ormore particles while allowing said one or more particles to continuetoward the at least one cell.
 6. An apparatus as in claim 1, whereinsaid means to accelerate comprises mechanical impulse means.